WO2016138563A1 - Nouveaux procédés de synthèse - Google Patents

Nouveaux procédés de synthèse Download PDF

Info

Publication number
WO2016138563A1
WO2016138563A1 PCT/AU2016/050140 AU2016050140W WO2016138563A1 WO 2016138563 A1 WO2016138563 A1 WO 2016138563A1 AU 2016050140 W AU2016050140 W AU 2016050140W WO 2016138563 A1 WO2016138563 A1 WO 2016138563A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
ligation
reaction
over
hplc
Prior art date
Application number
PCT/AU2016/050140
Other languages
English (en)
Inventor
Richard James Payne
Nicholas Joe MITCHELL
Lara Rebecca MALINS
Original Assignee
The University Of Sydney
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2015900739A external-priority patent/AU2015900739A0/en
Application filed by The University Of Sydney filed Critical The University Of Sydney
Priority to EP16758380.6A priority Critical patent/EP3265469A4/fr
Publication of WO2016138563A1 publication Critical patent/WO2016138563A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B19/00—Selenium; Tellurium; Compounds thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C07K1/026—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution by fragment condensation in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the disclosure relates to organic synthesis.
  • the disclosure relates to the synthesis of peptides and proteins.
  • the present disclosure relates to a method of preparing an amide containing compound comprising the step of reacting an acyl donor with a diselenide bearing an amino group.
  • the acyl donor is an acyl halide.
  • the acyl donor is an anhydride.
  • the acyl halide is an acyl bromide.
  • the acyl halide is an acyl chloride.
  • the present disclosure relates to a method of preparing an amide containing compound comprising the step of reacting an ester with a diselenide bearing an amino group. Preferably, the reaction proceeds in the absence of an additive.
  • the amide containing compound is preferably a peptide.
  • the peptide is defined by formula (I):
  • Nterm is the N-terminus of the peptide
  • Cterm is the C-terminus of the peptide
  • AA is an amino acid
  • n is an integer
  • (AA)n represents a peptide comprising n number of amino acid monomers
  • DG is a displaceable group
  • Preferab is Sec.
  • the DG is preferably a selenoate or a leaving group (LG).
  • the selenoate is an aryl selenoate.
  • the aryl selenoate is preferably phenyl selenolate.
  • the peptide is defined by formula (IV):
  • AAa AAb AAc AAd AAe ⁇ represents a peptide comprising the five amino acid residues AAa, AAb, AAc, AAd, and AAe.
  • AAa is L; AAb is Y; AAc is R; AAd is A; AAe is N.
  • X is preferably selected from the group consisting of: Ala, Ser, Thr, Leu, He, Val, Phe, Met and Lys.
  • AAu g is preferably U; AAi is S; AA 2 is P; AA is G; AA 4 is Y; AA 5 is S.
  • the reaction is conducted in an aqueous solution.
  • the aqueous solution has a pH in the range of about 2 to 14.
  • the aqueous solution has a pH in the range of about 2 to 8 when the reaction is run in the absence of an additive.
  • the pH can be in the range of about 8 to 14.
  • the aqueous solution is preferably a buffer comprising a denaturing agent and an aqueous solution of Na 2 HP0 4 .
  • the denaturing agent is 6 M guanidine hydrochloride.
  • the aqueous solution of Na 2 HP0 4 preferably has a concentration of about 100 mM.
  • the buffer is at a pH of about 7.2 when the reaction is run in the absence of an additive.
  • the ester and the diselenide are dissolved in the buffer before the reaction step at a concentration of about 10 mM.
  • the reaction is preferably commenced by combining the solutions of the ester and the diselenide and then allowed to proceed to completion as measured by an analytical technique.
  • the pH of the combined solution is at a pH of about 6.5 when the reaction is run in the absence of an additive and the final concentration of the reaction with respect to the ester peptide fragment 5 mM.
  • the reaction is preferably complete within about 60 seconds as measured by the consumption of the ester by HPLC excepting those reactions where X is selected from He or Val wherein the reaction is complete within about 10 minutes as measured by HPLC.
  • the method additionally comprises the step of deselenizing the peptide.
  • the peptide preferably comprises a cysteine residue and the deselenisation step comprises selectively deselenizing the peptide so as not to desulfurize the cysteine residue.
  • the deselenization preferably comprises reacting the peptide with a reducing agent.
  • the reducing agent may be a mild reducing agent.
  • the reducing agent comprises a phosphine.
  • the phosphine is preferably water soluble.
  • the phosphine is tris-(2-carboxyethyl)phosphine (TCEP).
  • the reducing agent preferably additionally comprises a thiol.
  • the thiol is dithiothreitol.
  • the deselenization preferably comprises reacting the peptide with a reducing agent and an oxidizing agent.
  • the reducing agent may be a mild reducing agent.
  • the oxidizing agent may be a mild oxidizing agent.
  • the reducing agent comprises a phosphine.
  • the phosphine is preferably water soluble.
  • the phosphine is tris-(2-carboxyethyl)phosphine (TCEP).
  • the oxidising agent is preferably potassium peroxy monosulfate.
  • the deselenization is conducted at a pH of about 4 to 5.
  • the reaction step and the deselenization step are preferably conducted in a one-pot reaction.
  • the present disclosure also relates to a method for oxidatively deselenizing a seleno functionalized amino acid residue in a peptide, said method comprising exposing the peptide to a mild reducing agent and a mild oxidizing agent.
  • the mild reducing agent preferably comprises a phosphine.
  • the phosphine is water soluble.
  • the phosphine is preferably tris-(2-carboxyethyl)phosphine (TCEP).
  • TCEP tris-(2-carboxyethyl)phosphine
  • the oxidizing agent is potassium peroxy monosulfate.
  • the deselenization is preferably conducted at a pH of about 4 to 5.
  • the disclosure also relates to a method of preparing an ester containing compound comprising the step of reacting an ester reagent with a diselenide bearing a hydroxyl group.
  • the ester reagent is a selenoester reagent.
  • the disclosure also relates to a method of preparing a hydrazide containing compound comprising the step of reacting an ester reagent with a diselenide bearing a hydrazine group.
  • the ester reagent is a selenoester reagent.
  • the disclosure also relates to a method of preparing an amide containing compound comprising the step of reacting an ester with a dithiol bearing an amino group, wherein the reaction proceeds in the absence of an additive.
  • the ester reagent is a selenoester reagent.
  • the disclosure also relates to a method of preparing an ester containing compound comprising the step of reacting an ester with a dithiol bearing a hydroxyl group, wherein the reaction proceeds in the absence of an additive.
  • the ester reagent is a selenoester reagent.
  • the disclosure also relates to a method of preparing a hydrazide containing compound comprising the step of reacting an ester with a dithiol bearing a hydrazine group, wherein the reaction proceeds in the absence of an additive.
  • the ester reagent is a selenoester reagent.
  • the disclosure also relates to a method for preparing a phenylselenoester, the method comprising the step of treating a carboxylic acid compound with diphenyldiselenide (DPDS) followed by Bu 3 P.
  • DPDS diphenyldiselenide
  • the selenoester is a peptide selenoester and the carboxylic acid is a peptide carboxylic acid.
  • the disclosure also relates to a peptide of Formula (I) as described above.
  • additive refers to any means for promoting a reaction and/or preventing a side reaction that is added separately, from an external source, to a reaction mixture.
  • the reaction mixture contains reagents.
  • the reaction mixture may include a solvent.
  • the solvent may include an aqueous solution.
  • the aqueous solution may include buffering salts and a denaturing agent.
  • an additive may refer to an exogenous molecule that is added to the reaction mixture.
  • An additive may also refer to the addition of electrons as required in an electrochemical reduction.
  • Some non-limiting examples of additives are nucleophiles and reductants.
  • thiol group containing reductants that have been traditionally used in NCL are MPAA, thiophenol and MESNa.
  • Reductants that may be used with the selenocystine- selenoester ligation methodology disclosed herein include, but are not limited to, TCEP (tris(2-carboxyethyl)phosphine), THPP (tris(3-hydroxypropyl)phosphine), DTT (dithiothreitol), NaBH 4 , NaHBH 3 CN and ascorbic acid.
  • TCEP tris(2-carboxyethyl)phosphine
  • THPP tris(3-hydroxypropyl)phosphine
  • DTT dithiothreitol
  • NaBH 4 NaHBH 3 CN
  • ascorbic acid ascorbic acid.
  • selenol-based nucleophiles and reductants may also be used.
  • Additives to suppress side reactions such as deselenization during ligation include, but are not limited to, diphenyldiselenide (DPDS) or ascorbic acid or a salt thereof.
  • DPDS diphenyldiselenide
  • the methods disclosed herein may be performed in the absence of an additive.
  • the additive is ascorbic acid or a salt thereof.
  • the additive is sodium ascorbate.
  • amino acid refers to a molecule containing both an amino group and a carboxy group.
  • a-amino group attached directly to the carbon atom bearing both an amino and a carboxyl group
  • a-carboxyl group attached directly to the carbon atom bearing both an amino and a carboxyl group.
  • carboxyl may refer to either a -COOH group or a -COO " group
  • oc-amino acids are of the general form 3 ⁇ 4N- CHR-COOH, where R is a side chain or H.
  • the side chain in general is an alkyl chain, which is optionally substituted, commonly but not necessarily at its distal end.
  • the N terminus of the amino acid (or of a peptide) is that end at which the amine functionality (optionally ionised or substituted/protected) is located, and the C terminus is the end at which the carboxyl functionality (optionally ionised or substituted/protected) is located.
  • peptide refers to a chain comprising (or consisting of) at least two amino acid residues joined by amide bond(s). They may be dipeptides, oligopeptides, polypeptides, proteins, glycopeptides, glycoproteins etc.
  • peptide polypeptide and protein are used interchangeably herein and include a molecular chain of two or more amino acids linked covalently through peptide bonds. The terms do not refer to a specific length of the product.
  • the terms include post- translational modifications of the peptide, for example, glycosylations, acetylations, biotinylations, 4-pentynoylations, PEGylations, phosphorylations, sulfations and the like.
  • protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide.
  • the terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included.
  • peptides can be derivatized as described herein by well-known organic chemistry techniques set forth, for example, in Smith, M. B.
  • aryl alone or in combination, means a carbocyclic aromatic moiety containing one, two or even three rings wherein such rings may be attached together in a fused manner.
  • aryl embraces aromatic radicals such as phenyl, naphthyl, indenyl, tetrahydronaphthyl, anthracenyl, and indanyl.
  • Said "aryl” group may have 1 or more substituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro, cyano, alkoxy and lower alkylamino, and the like. Phenyl substituted with -O- CH 2 -O- forms an aryl benzodioxolyl substituent.
  • Aryl as used herein, implies a fully unsaturated ring.
  • Groups that are displaceable generally refer to groups that are displaceable from a molecule during the course of a reaction.
  • leaving groups generally refer to groups that are displaceable by a nucleophile. Such leaving groups are known in the art. Examples of leaving groups include, but are not limited to, halides (e.g., I, Br, F, CI), sulfonates (e.g., mesylate, tosylate), sulfides (e.g., SCH 3 ), thiolate, selenoates, N-hydroxysuccinimide, N- hydroxybenzotriazole, and the like.
  • halides e.g., I, Br, F, CI
  • sulfonates e.g., mesylate, tosylate
  • sulfides e.g., SCH 3
  • thiolate thiolate
  • selenoates N-hydroxysuccinimide
  • N- hydroxybenzotriazole and the like.
  • Nucleophiles are species that are capable of attacking a molecule at the point of attachment of the leaving group causing displacement of the leaving group. Nucleophiles are known in the art. Examples of nucleophilic groups include, but are not limited to, amines, thiols, alcohols, selenols, Grignard reagents, anionic species (e.g., alkoxides, amides, carbanions) and the like.
  • Non-canonical or non-proteogenic amino acid residues can be incorporated into a peptide by employing the techniques disclosed herein.
  • the term "non-canonical amino acid residue” refers to amino acid residues in D- or L-form that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins, for example, ⁇ -amino acids, homoamino acids, cyclic amino acids, seleno amino acids, thio amino acids, and amino acids with derivatized side chains such as those described in US 2015/0023988.
  • the peptides described can also be chemically derivatized at one or more amino acid residues by known organic chemistry techniques.
  • “Derivative” or “derivatized” refers to a subject peptide having one or more residues chemically derivatized by reaction of a functional side group.
  • Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-benzylhistidine. Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty canonical amino acids, whether in L- or D-form.
  • 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
  • Useful derivatizations include modification of an N-terminal free amino group for attachment of an imaging agent, e.g. a fluorescent dye or a therapeutic agent whose activity adds to the potential therapeutic activity of the peptide.
  • the N-terminus can be acylated or modified to a substituted amine, or derivatized with another functional group, such as an aromatic moiety (e.g., an indole acid, benzyl (Bzl or Bn), dibenzyl (DiBzl or Bn 2 ), or benzyloxycarbonyl (Cbz or Z)), ⁇ , ⁇ -dimethylglycine or creatine.
  • an imaging agent e.g. a fluorescent dye or a therapeutic agent whose activity adds to the potential therapeutic activity of the peptide.
  • the N-terminus can be acylated or modified to a substituted amine, or derivatized with another functional group, such as an aromatic moiety (e.g., an indole acid,
  • an acyl moiety such as, but not limited to, a formyl, acetyl (Ac), propanoyl, butanyl, pentanyl, heptanyl, hexanoyl, octanoyl, or nonanoyl, can be covalently linked to the N-terminal end of the peptide.
  • N-terminal derivative groups include -NRRi (other than -NH 2 ), -NRC(0)Ri, -NRC(0)ORi, -NRS(0) 2 Ri, -NHC(0)NHRi, succinimide, or benzyloxycarbonyl-NH- (Cbz-NH-), wherein R and Ri are each independently hydrogen or lower alkyl or phenyl and wherein the phenyl ring may be substituted with 1 to 5 substituents selected from Ci-C 4 alkyl, Ci-C 4 alkoxy, chloro, and bromo.
  • one or more peptidyl [-C(0)NR-] linkages (bonds) between amino acid residues can be replaced by a non-peptidyl linkage.
  • exemplary non-peptidyl linkages are -CH 2 -carbamate [-CH 2 -OC(0)NR-], phosphonate, -CH 2 - sulfonamide [-CH2-S(0) 2 NR-], thiourea [-NHC(S)NH-], urea [-NHC(0)NH-], -CH 2 - secondary amine, and alkylated peptide [-C(0)NR 6 , wherein R 6 is lower alkyl] .
  • Figure 1 Generalised synthetic scheme for preparation of amides, esters and hydrazides using the ligation and selenium chemistries disclosed herein.
  • Figure 2. General Scheme showing the solid-phase synthesis of peptide selenoesters and amino peptide ligation intermediates; additive free ligation; and selective reductive and oxidative deselenizations to afford peptides with Alanine and Serine at the ligation site.
  • Selenocystine is the oxidised (diselenide) form of selenocysteine (Sec).
  • FIG. 5 Scheme showing additive-free ligation of a model selenoester (Ac- LYRANA-SePh, 2) and selenocystine-containing dimer (H-USPGYS-NH 2 ) 2 1 in aqueous, denaturing buffer at neutral pH to afford Ac-LYRANAUSPGYS-NH 2 symmetrical dimer 11a and unsymmetrical diselenide product lib.
  • Ac- LYRANA-SePh 2
  • H-USPGYS-NH 2 selenocystine-containing dimer
  • Figure 7 Scheme of one-pot Ac-LYRANA-SePh + H-USPGYS-NH 2 dimer ligation followed by Sec to Ala and Sec to Ser conversion.
  • Figure 8. Synthesis of a peptide with Ser at the ligation site using a selective oxidative deselenization.
  • Figure 10 A selenoester ligation with a 2-thiol tryptophan-containing peptide.
  • Figure 13 Generalised synthetic scheme for the preparation of peptide on solid support using selenocystine- selenoester ligation.
  • Figure 14 Generalised solid-phase synthesis of Ac-LYRANAUSPGYS-NH 2 (11) using selenocystine- selenoester ligation chemistry.
  • Figure 16 One pot ligation-deselenization using a model sequence containing cysteine.
  • Figure 18 Progress plot of the ligation reaction between Ac-LYRANL-SeAlk (51) and H-USPGYS-NH 2 dimer (1) at 2.5 mM with respect to 1 over the course of 12 h.
  • Figure 19 Ligation reaction between Ac-LYRANP-SePh (52) and H- USPGYS-NH 2 dimer (1).
  • Figure 21 Ligation reaction between Ac-LYRANI-SePh (6) and H- USPGYS-NH 2 dimer (1) at 2.5 mM with respect to peptide 1 in the presence of 200 mM TCEP.
  • FIG. 26 Scheme depicting the ligation reaction of H-USPGYS-NH 2 dimer (1) with Ac-LYRANL-SePh (5) and the ligation reaction of H-USPGYS-NH 2 dimer (1) with Ac-LYRANL-SePhR (54 to 59).
  • Figure 40 Preparation of 12 by ligation reaction between Ac-LYRANS-SePh + H-USPGYS-NH 2 dimer.
  • Figure 43 Preparation of Ac-LYRANTUSPGYS-NH 2 (13) by ligation reaction between Ac-LYRANT-SePh + H-USPGYS-NH 2 dimer.
  • Figure 59 Ligation reaction between H-USPGYS-NH 2 dimer and Ac- LYRANM-SePh to give 18a-c.
  • FIG. 60 Ligation reaction between H-USPGYS-NH2 dimer and Ac- LYRANK-SePh to give 19a-c.
  • Analytical UPLC trace of HPLC purified ligation product Ac-LYRANKUSPGYS-NH 2 (19a); Rt 17.0 min (0-50% B over 30 min, ⁇ 214 nm); Calculated Mass [M+3H] 3+ : 964.7 (100%), [M+4H] 4+ : 723.8 (100%); Mass Found (ESI + ); 964.5 [M+3H] 3+ , 723.9 [M+4H] 4+ .
  • Figures 75 to 78 show the ligation of Ac-LYRANT-SePh + H-USPGYS-NH 2 dimer and the conversion of the Sec residue at the ligation site to provide Alanine and Serine residues at the ligation site.
  • FIG. 75 The ligation of Ac-LYRANT-SePh + H-USPGYS-NH 2 and the conversion of the Sec residue at the ligation site to provide Alanine and Serine residues at the ligation site.
  • FIG. 123 Circular dichroism spectrum of native Mtb CM 36 (250-195) in 50 mM Tris, 0.1 M NaCl, pH 7.5. (Data interval: 1 nm, bandwidth: 1.00 nm, scanning speed: 20 nm/min, accumulations: 5).
  • Figure 138 Circular dichroism spectrum of native ESAT-6 40 (250-195) in 25 mM NaH 2 P0 4 , 0.1 M NaCl, pH 6.5. (Data interval: 1 nm, bandwidth: 1.00 nm, scanning speed: 20 nm/min, accumulations: 5).
  • FIG. 139 Synthesis of H-USPCYS-NH 2 dimer 49.
  • Analytical HPLC: Rt 13.7 (intramolecular selenyl- sulfide, 49a), 16.5 min (diselenide dimer, 49b) (0-25% B over 30 min, ⁇ 230 nm); Calculated Mass (intramolecular selenyl- sulfide) [M+H] + : 704.2 (100%); Mass Found (ESI + ); 704.3 [M+H] + ; Calculated Mass (diselenide dimer) [M+H] + : 705.2 (100%); Mass Found (ESI + ); 706.3 [M+H] + .
  • Figure 142 HPLC trace of a ligation reaction between Ac-LYRANL-SeAlkyl
  • Figure 201 Mass data for compound 75. Calculated Mass [M+2H] + : 1500.6 (100%), [M+3H] 3+ : 1000.7 (100%); Mass Found (ESI+): 1500.6 [M+2H] 2+ , 1000.6 [M+3H] 3+ .
  • Figure 208 Synthetic route towards Boc-(p-PMBSe)Asp-OH
  • Figure 212 One-pot ligation-deselenisation of model peptide systems; [a] 0.5 eq. of H-(p-Se)LSPGYS-NH 2 diselenide dimer to 1.28 eq. of selenoester.
  • the disclosure relates to the surprising discovery that a peptide carrying an N- terminal selenocystine amino acid residue can be chemo selectively ligated to a peptide fragment bearing a C-terminal selenoester-functionalized amino acid residue.
  • the reaction proceeds without any additive - no reductant is required to reduce the selenocystine to free selenocysteine, no nucleophilic thiol is required, no exogenous nucleophilic selenol is required.
  • the reaction proceeds quickly using unprotected peptide fragments, in aqueous buffer with broad pH range at room temperature.
  • the reaction may be conducted at moderately elevated temperatures, or at room temperature or below.
  • the ligation reactions described herein are conducted at room temperature. Nonetheless, the skilled addressee would understand that the reactions can be run at a lower temperature to minimise side reactions or run at elevated temperatures to, for example, further accelerate the rate of reaction.
  • Suitable lower temperatures are below room temperature, below 0°C down to about -100 °C; for example, -10, -20, -50 or -70°C, or about -100 to about 0°C, or about -100 to -50, -100 to -70, -50 to 0, -20 to 0 or -80 to -60°C, e.g.
  • Suitable elevated temperatures are above room temperature, above about 30, 40, 50, 60, 70, up to about 80 °C.
  • the reaction may be run in the presence of an additive where the pH of the reaction mixture ranges from about 2 to 14. It would be understood, for example, that the reaction may be run in the presence of an additive at a pH from about 2 to 14; 2 to 13; 2 to 12; 2 to 11; 2 to 10; 2 to 9; 2 to 8; 2 to 7; 2 to 6; 2 to 5; 2 to 4; 2 to 3; 3 to 14; 3 to 13; 3 to 12; 3 to 11; 3 to 10; 3 to 9; 3 to 8; 3 to 7; 3 to 6; 3 to 5; 3 to 4; 4 to 14; 4 to 13; 4 to 12; 4 to 11; 4 to 10; 4 to 9; 4 to 8; 4 to 7; 4 to 6; 4 to 5; 5 to 14; 5 to 13; 5 to 12; 5 to 11; 5 to 10; 5 to 9; 5 to 8; 5 to 7; 5 to 6; 6 to 14; 6 to 13; 6 to 12; 6 to 12; 6 to 11; 6 to 10; 6 to 9; 6 to 8; 6 to 13
  • the reaction may be run in the absence of an additive at a pH from about 2 to 8; 2 to 7; 2 to 6; 2 to 5; 2 to 4; 2 to 3; 3 to 8; 3 to 7; 3 to 6; 3 to 5; 3 to 4; 4 to 8; 4 to 7; 4 to 6; 4 to 5; 5 to 8; 5 to 7; 5 to 6; 6 to 8; 6 to 7; or 7 to 8.
  • the reaction may be run, for example, at a pH of about 2.0 or 2.1 or 2.2 or 2.3 or 2.4 or 2.5 or 2.6 or 2.7 or 2.8 or 2.9 or 3 or 3.1 or 3.2 or 3.3 or 3.4 or 3.5 or 3.6 or 3.7 or 3.8 or 3.9 or 4 or 4.1 or 4.2 or 4.3 or 4.4 or 4.5 or 4.6 or 4.7 or 4.8 or 4.9 or 5 or 5.1 or 5.2 or 5.3 or 5.4 or 5.5 or 5.6 or 5.7 or 5.8 or 5.9 or 6 or 6.1 or 6.2 or 6.3 or 6.4 or 6.5 or 6.6 or 6.7 or 6.8 or 6.9 or 7 or 7.1 or 7.2 or 7.3 or 7.4 or 7.5 or 7.6 or 7.7 or 7.8 or 7.9 or 8.0.
  • the reactions may be carried out using conventional heating or microwave irradiation or with flow chemistry performed in a fluidic device e.g. a micro-fluidic reactor.
  • the reactions may be conducted under an inert atmosphere, e.g. nitrogen, helium, argon, carbon dioxide etc.
  • an equivalent molar ratio most of our selenoester examples proceeded to completion at a concentration of 1 mM (notable exceptions are the Val and lie esters which necessitate 1.25 eq). At 2 molar equivalents the ligations could be effectively run at 500 ⁇ . Below this concentration the reaction stalled.
  • the ligation and selective deselenization steps described above may conveniently be conducted as a one-pot reaction. They may be conducted without isolation or purification of intermediate species. Thus, following the ligation reaction, the crude reaction mixture may be subjected, without purification of intermediates (but optionally with at least partial removal of at least one reagent or catalyst used in the ligation reaction), to suitable deselenization conditions and reagents. The resulting ligated and selectively deselenized product peptide may be obtained from the resulting reaction mixture following a suitable time for reaction.
  • Chorismate Mutase is an enzyme isolated from Mycobacterium tuberculosis that catalyzes the conversion of chorismate to prephenate, a key intermediate in the biosynthesis of tyrosine (Tyr) and phenylalanine (Phe).
  • our synthetic folded enzyme had similar structure and activity to that reported for the recombinant protein (Prakash, P.; Aruna, B.; Sardesai, A. A.; Hasnain, S. E. J. Biol. Chem. 2005, 280, 19641 and Kim, S.-K.; Reddy, S. K.; Nelson, B. C; Robinson, H.; Reddy, P. T.; Ladner, J. E. FEBS Journal 2008, 275 4824) as determined by circular dichroism (CD) spectroscopy and by a kinetic assay with chorismate.
  • CD circular dichroism
  • ESAT-6 N-aceylated Cys-free 94 residue protein early secretory antigenic protein-6
  • Figure 12 ESAT-6, also from Mtb, is an important virulence factor and a potent T cell antigen (Sorensen, A. L.; Nagai, S.; Houen, G.; Andersen, P.; Andersen, A. B. Infect. Immun. 1995, 63, 1710).
  • ESAT-6 1-39 as an N-terminal phenylselenoester
  • ESAT-6 40-71 dimer 38) containing an N-terminal selenocystine moiety and C-terminal alkyl thioester
  • ESAT-6 72-94 319 which we proposed to assemble via a one-pot, three-component ligation reaction using both native chemical ligation and the additive-free selenocystine-selenoester ligation methodology.
  • the ligation was allowed to proceed at 37 °C for 16 h, which led to completion of the native chemical ligation reaction together with concomitant deselenization of Sec-40 to Ala due to the addition of TCEP (Metanis, N.; Keinan, E.; Dawson, P. E. Angew. Chem. Int. Ed. 2010, 49, 7049).
  • the reaction mixture was subsequently dosed with the water- soluble radical initiator VA-044, (Wan, Q.; Danishefsky, S. J. Angew. Chem. Int. Ed.
  • the present disclosure also contemplates the deployment of the selenium chemistries disclosed herein so that they are amenable to solid-phase synthesis of molecules, for example, peptides.
  • the majority of protein targets (with or without modifications) are >85 amino acids in length and, due to the size limitations of solid-phase peptide synthesis (SPPS), require the ligation of three or more peptide fragments for assembly.
  • SPPS solid-phase peptide synthesis
  • each individual ligation step requires purification by reverse-phase HPLC before proceeding to the next ligation (or deprotection) reaction, inevitably leading to the use of large quantities of solvent and significant handling losses.
  • each purification step entails time-consuming lyophilization procedures (ca. 24 h) to enable solvent exchange for subsequent reactions.
  • Preparative reverse-phase HPLC was performed using a Waters 600 Multisolvent Delivery System and Waters 500 pump with 2996 photodiode array detector or Waters 490E Programmable wavelength detector operating at 230 and 254 nm.
  • Peptides were purified on a Waters Sunfire 5 ⁇ (C-18) preparative column operating at a flow rate of 7 mL min "1 or an XBridge BEH 5 ⁇ wide-pore (C-18) using a mobile phase of 0.1% trifluoroacetic acid in water (Solvent A) and 0.1% trifluoroacetic acid in acetonitrile (Solvent B) and a linear gradient of 0-50% B over 40 min.
  • LC-MS was performed either on a Shimadzu LC-MS 2020 instrument consisting of a LC-M20A pump and a SPD-20A UV/Vis detector coupled to a Shimadzu 2020 mass spectrometer (ESI) operating in positive mode or a Shimadzu UPLC-MS equipped with the same modules as the LC-MS system except for a SPD- M30A diode array detector. Separations were performed on the LC-MS system either on a Waters Sunfire 5 ⁇ , 2.1 x 150 mm column (C-18), or wide-pore equivalent operating at a flow rate of 0.2 mL min "1 .
  • ESI Shimadzu 2020 mass spectrometer
  • Separations on the UPLC-MS system were performed using a Waters Acquity UPLC BEH 1.7 ⁇ 2.1 x 50 mm column (C-8) at a flow rate of 0.6 mL min "1 . Separations were performed using a mobile phase of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) and a linear gradient of 0-50% B over 30 min.
  • the pale-gray Grignard solution was transferred via canula to a clean, flame-dried flask under an atmosphere of argon.
  • the solution was cooled to 0 °C and treated with selenium powder (1.0 eq.).
  • the reaction mixture was warmed to room temperature and stirred for 16 h.
  • the crude reaction mixture was poured into saturated aqueous NH 4 C1 (25 mL) and extracted with EtOAc (3 x 20 mL). The combined organic extracts were dried (MgS0 4 ), filtered and concentrated in vacuo.
  • the crude residue was eluted through a silica plug (0: 100 to 20:80 EtO Ac/Hex) to provide the aryl diselenide.
  • Method B Aryldiazonium approach [354] To a solution of / ⁇ -substituted aniline (50 mg) in 10% (v/v) aq. HCl/MeOH (0.5 mL) at 0 °C was added dropwise a solution of NaN0 2 (1.1 eq.) in H 2 0 (0.5 mL). The reaction mixture was stirred at 0 °C for 30 min. The resulting diazonium salt was added to a solution of selenourea (2 eq.) and CuCl 2 (0.25 eq.) in 10% (v/v) H 2 0/MeOH (0.5 mL).
  • the resulting mixture was stirred at 35 °C for 2 h, cooled to room temperature and then extracted with CHC1 3 .
  • the aqueous layer was concentrated via lyophilization.
  • the crude residue was resuspended in methanol (2 mL) and treated with excess 30% aqueous NH 4 OH (0.5 mL).
  • the reaction mixture was heated at 50 °C for 1 h.
  • the crude reaction mixture was acidified with 1 M HC1 and extracted with EtOAc (3 x 20 mL).
  • the combined organic layers were dried (MgS0 4 ), filtered and concentrated in vacuo.
  • the crude product was then purified by flash column chromatography through a silica plug or via reverse-phase HPLC.
  • Method B is a modification of the procedure published by Stuhr-Hansen and coworkers, in which an aryldiazonium species is reacted with KSeCN in the presence of NaOAc to form an arylselenocyanate. The selenocyanate may then be converted to the corresponding diselenide upon treatment with H 2 S0 4 and 0 2 .
  • selenourea serves as the nucleophilic selenium species in place of KSeCN. Note that the addition of CuCl 2 with selenourea is based on literature precedent for the arylation of thiourea with aryldiazonium salts (Kopylova, B. V. et ah, Russ. Chem. Bull. 1973, 22, 2663 (page 2664, paragraph 6)).
  • Method C Aryldiazonium approach using commercially available aryldiazonium salts.
  • Method B can be carried out with commercially available aryldiazonium salts (e.g. 4-nitrobenzenediazonium tetrafluoroborate).
  • aryldiazonium salts e.g. 4-nitrobenzenediazonium tetrafluoroborate
  • a solution of commercially available diazonium salt (-100 mg) was treated directly with selenourea and CuCl 2 .
  • Rink amide resin was initially washed with DCM (5 x 3 mL) and DMF (5 x 3 mL), followed by removal of the Fmoc group by treatment with 20% piperidine/DMF (2 x 5 min). The resin was washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL). PyBOP (4 eq.) and NMM (8 eq.) were added to a solution of Fmoc-AA- OH (4 eq.) in DMF. After 5 min of pre-activation, the mixture was added to the resin.
  • the resin was treated with a solution of DCM/CH30H/z ' Pr 2 NEt (17:2: 1 v/v/v) for 1 h and washed with DMF (5 x 3 mL), DCM (5 x 3 mL), and DMF (5 x 3 mL). The resin was subsequently submitted to iterative peptide assembly (Fmoc-SPPS).
  • Model peptide selenoesters were prepared on 2-chlorotrityl chloride resin using Fmoc-SPPS as described in the general methods. Cleavage of the peptides from the resin was effected by treating with 30 vol.% HFIP in DCM for 2 h before concentrating in vacuo. The resulting residue was dissolved in anhydrous DMF and cooled to 0 °C. Diphenyl diselenide (30 eq. in DMF) was added to the solution followed by Bu 3 P (30 eq.). The reaction was allowed to proceed at 0 °C for 3 h, after which time the solvent was removed in vacuo.
  • Each model ligation was analysed at appropriate time points (60, 90, 300 and/or 600 seconds) via direct inject HPLC with a gradient of 0-60% B over 30 mins using a Waters Sunfire 5 ⁇ 4.6 x 250 mm (C-18) column at a flow rate of 2 mL/min.
  • 40 ⁇ ⁇ of the crude ligation solution was diluted up to 1 mL with 1 % TFA/H 2 0 and injected into the HPLC loop.
  • the peptide solution was treated with the TCEP solution and the DTT solution simultaneously to give a final concentration of 2.5 mM with respect to the peptide ligation product and a final reaction pH of 4.5-5.
  • the solution was agitated on an orbital shaker at rt and monitored by LC-MS analysis. If necessary, additional aliquots of aqueous TCEP and DTT were added.
  • the solution was purified via semi-preparative reverse-phase HPLC employing a mobile phase of 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile (Solvent B) with a linear gradient as specified. All peptide products were isolated as white solids following lyophilization.
  • the solution was agitated on an orbital shaker at rt and monitored by LC-MS analysis. If necessary, additional aliquots of aqueous TCEP (pH adjusted to 7.5-7.7) and aqueous Oxone were added. Following completion of the reaction, the solution was concentrated on a lyophilizer (16 h). The samples were reconstituted in water containing 0.1% TFA and centrifuged. The supernatant was collected and purified via semi-preparative reverse-phase HPLC employing a mobile phase of 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile (Solvent B) with a linear gradient as specified. All peptide products were isolated as white solids following lyophilization.
  • the peptide ligation product (1.5-2.5 mg) was dissolved in buffer (6 M guanidine hydrochloride, 100 mM Na 2 HP0 4 , adjusted to pH 7.5, 5 mM with respect to the ligation product). The peptide was diluted to a concentration of 200 ⁇ by the addition of distilled water. A solution of TCEP (50 eq.) in water (15 mM) was prepared and adjusted to a final pH of 7.5-7.7 with 2 M NaOH. A solution of Oxone (50 eq.) in water (30 mM) was also prepared.
  • the peptide solution was treated simultaneously with the aqueous solutions of TCEP and Oxone to give a final concentration of 100 ⁇ with respect to the peptide ligation product and a final reaction pH of 4.2-4.5.
  • the solution was agitated on an orbital shaker at rt and monitored by LC-MS analysis. If necessary, additional aliquots of aqueous TCEP (pH adjusted to 7.5-7.7) and aqueous Oxone were added.
  • crude peptide products were concentrated on a lyophilizer (16 h). The samples were reconstituted in water containing 0.1% TFA and centrifuged.
  • the supernatant was collected and purified via semi-preparative reverse-phase HPLC employing a mobile phase of 0.1% TFA in water (Solvent A) and 0.1% TFA in acetonitrile (Solvent B) with a linear gradient as specified.
  • Peptide 41 was synthesized from the corresponding Sec peptide ligation product (1.8 mg, 1.2 ⁇ ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 41 as a white solid (1.3 mg, 83% yield). Analytical data is shown in Figure 82 and Figure 83.
  • Peptide 29 was synthesized from selenyl-MPAA sulfide peptide ligation product (2.5 mg, 1.6 ⁇ ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 29 as a white solid (1.9 mg, 90% yield). Analytical data is shown in Figure 84 and Figure 85.
  • Peptide 43 was synthesized from the selenyl-MPAA sulfide peptide ligation product (1.5 mg, 1.0 ⁇ ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 43 as a white solid (1.3 mg, 93% yield). Analytical data is shown in Figure 89 and Figure 90.
  • Peptide 45 was synthesized from the selenyl-MPAA sulfide peptide ligation product (1.7 mg, 1.0 ⁇ ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 40% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 45 as a white solid (1.3 mg, 93% yield). Analytical data is shown in Figure 93 and Figure 94.
  • Peptide 46 was synthesized from the selenyl-MPAA sulfide peptide ligation product (1.7 mg, 1.1 ⁇ ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Purification of the concentrated reaction mixture via semi-preparative reverse phase HPLC (0 to 40% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 46 as a white solid (1.4 mg, 95% yield). Analytical data is shown in Figure 95 and Figure 96.
  • Peptide 47 was synthesized from a mixture of the diselenide dimer ligation product (1.0 mg, 0.62 ⁇ ) and the corresponding selenyl-MPAA sulfide ligation product (1.0 mg, 0.64 ⁇ ) according to the optimized Sec to Ser conversion protocol outlined in the general methods (reaction condition B). Direct purification of the crude (unconcentrated) reaction mixture via semi-preparative reverse phase HPLC (0 to 50% B over 40 min, 0.1% TFA) followed by lyophilization afforded peptide 47 as a white solid (1.5 mg, 80% yield). Analytical data is shown in Figure 97 and Figure 98.
  • Reaction monitoring via HPLC-MS analysis at t 6 h (0 to 50% B over 30 min) indicated consumption of the ⁇ -selenophenylalanine-containing peptide and formation of ligated peptide products as a mixture of the diselenide dimer peptide, the asymmetric phenylselenyl diselenide and the trans-esterified internal selenoester product (see Figures 102-104).
  • Mtb CM (36) activity assays were carried out as described by Davidson and Hudson (Davidson, B. E., and Hudson, G. S. (1987) Methods Enzymol. 142, 440-450). Briefly, to 200 ⁇ ⁇ of chorismic acid solution in 50 mM Tris-HCl, 10 mM mercaptoethanethiol, 0.1 mg/mL BSA, pH 7.5 (1, 0.5, 0.25 & 0.125 mM) incubated for 5 min at 37 °C was added 10 of Mtb CM (10 ⁇ solution; 100 pmol). The reactions were then incubated to their desired time point (2.5, 10, 20, 30, 60, 120 or 180 min) and quenched with 200 1 M HC1.
  • the samples were further incubated for 10 min before addition of 400 ⁇ ⁇ 2.5 M NaOH and analysis by UV/Vis at 320 nm using a control sample minus addition of the Mtb CM enzyme as a blank.
  • the assays are depicted in Figures 124 to 128.
  • the ligation was allowed to proceed at 37 °C overnight. After this time 29 mg TCEP (0.1 mmol) and 6.14 mg glutathione (0.02 mmol) were dissolved into 480 ⁇ ⁇ ligation buffer and the pH adjusted to 7.5. The ligation solution was degassed, treated with the solution of TCEP and glutathione, and 3.23 mg VA-044 (0.01 mmol) were added to the combined reaction mixture as a solid. The reaction was allowed to proceed at 37 °C overnight.
  • H-USPCYS-NH 2 dimer was synthesized using Fmoc-strategy SPPS on Rink amide resin (25 ⁇ ) through the direct incorporation of (Boc-Sec-OH) 2 as outlined in the general methods section.
  • the crude peptide was purified by preparative reverse- phase HPLC (0 to 30% B over 40 min, 0.1% TFA) and lyophilized to afford the desired peptide as a mixture of the intramolecular selenyl-sulfide (49a) and diselenide dimer (49b) (9.0 mg, 51% yield).
  • Analytical data is shown in Figure 139.
  • the resin was washed with buffer (6 M guanidine hydrochloride, 0.1 M Na 2 HP0 4 , 3 x 3 mL), followed by H 2 0 (3 x 3 mL), DMF (3 x 3mL) and DCM (10 x 3 mL).
  • the peptide was then cleaved from the resin upon treatment with a solution of TFA/z ' Pr 3 SiH/H 2 0 (90:5:5 v/v/v, 2 mL, 2 h) and the cleavage solution was subsequently concentrated under a stream of nitrogen.
  • Rink amide Chem-Matrix resin was initially washed with DCM (5 x 3 mL) and DMF (5 x 3 mL), followed by removal of the Fmoc group by treatment with 20% piperidine/DMF (2 x 5 min). The resin was then washed with DMF (5 x 3 mL), DCM (5 x 3 mL) and DMF (5 x 3 mL). PyBOP (4 eq.) and NMM (8 eq.) were added to a solution of (Fmoc-Sec-OH) 2 (2 eq.) in DMF. After 5 min of pre-activation, the mixture was added to the resin.
  • the reaction was allowed to proceed at 0 °C for 3 h, after which time the solvent was removed in vacuo.
  • the crude material was put on ice and the protecting groups removed via treatment with TFA:TIS:thioanisole:H 2 0 (85:5:5:5 v/v/v/v).
  • the cleavage cocktail was removed under a stream of N 2 and the crude residue suspended in diethyl ether and cooled to -20 °C.
  • the precipitate was pelleted by centrifugation at 4000 rpm for 5 min.
  • Fmoc-Ser-OH was loaded to 2-chlorotrityl chloride ChemMatrix resin (150 ⁇ ) and the peptide was elongated using automated Fmoc-SPPS as outlined in the general procedures. After coupling of Boc-(P-PmbSe)Asp-OH to the N-terminus, the fully protected resin-bound peptide was cleaved and deprotected using a solution of TFA/z ' Pr 3 SiH/H 2 0 (89:5:5 v/v/v). The solution was agitated at room temperature for 2 h and then concentrated in vacuo. Crude peptide was precipitated from cold Et 2 0, centrifuged, and used directly.
  • the reaction was allowed to proceed at 0 °C for 3 h, after which time the solvent was removed in vacuo.
  • the crude material was put on ice and the protecting groups removed via treatment with TFA:TIS:thioanisole:H 2 0 (85:5:5:5 v/v/v/v).
  • the cleavage cocktail was removed under a stream of N 2 and the crude residue suspended in diethyl ether and cooled to -20 °C.
  • the precipitate was pelleted by centrifugation at 4000 rpm for 5 min.
  • Fmoc-Ser-OH was loaded to 2-chlorotrityl chloride ChemMatrix resin (150 ⁇ ) and the peptide was elongated using automated Fmoc-SPPS as outlined in the general procedures. After coupling of Boc-(P-PmbSe)Asp-OH to the N-terminus, the fully protected resin-bound peptide was cleaved and deprotected using a solution of TFA/z ' Pr 3 SiH/H 2 0 (89:5:5 v/v/v). The solution was agitated at room temperature for 2 h and then concentrated in vacuo. Crude peptide was precipitated from cold Et 2 0, centrifuged, and used directly.
  • Hyalomin-4 (26-51) dimer (2.47 mg, 0.46 umol) and Hyalomin-3 (1-25) selenoester (4.49 mg, 1.497 ⁇ ) together with in situ deselenization was performed as outlined in the general methods section. Purification via preparative reverse phase HPLC (0 to 60% B over 30 min, 0.1% TFA) followed by lyophilization afforded the native Hyalomin-4 protein (77) (3.3 mg, 66%) as a white solid. Analytical data shown in Figures 204 to 207.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne de nouveaux procédés de synthèse organique. L'invention concerne notamment la synthèse rapide de peptides et de protéines.
PCT/AU2016/050140 2015-03-03 2016-03-03 Nouveaux procédés de synthèse WO2016138563A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP16758380.6A EP3265469A4 (fr) 2015-03-03 2016-03-03 Nouveaux procédés de synthèse

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
AU2015900739A AU2015900739A0 (en) 2015-03-03 New synthetic methods
AU2015900739 2015-03-03
AU2015902928A AU2015902928A0 (en) 2015-07-23 New synthetic methods
AU2015902928 2015-07-23

Publications (1)

Publication Number Publication Date
WO2016138563A1 true WO2016138563A1 (fr) 2016-09-09

Family

ID=56849119

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2016/050140 WO2016138563A1 (fr) 2015-03-03 2016-03-03 Nouveaux procédés de synthèse

Country Status (2)

Country Link
EP (1) EP3265469A4 (fr)
WO (1) WO2016138563A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114249794A (zh) * 2021-12-22 2022-03-29 深圳瑞德林生物技术有限公司 一种氧化型谷胱甘肽的合成方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5948418A (en) * 1996-03-29 1999-09-07 Estee Lauder, Inc. Sulfur-based amides and bis-amides useful against skin disorders
WO2010111737A1 (fr) * 2009-03-31 2010-10-07 The University Of Sydney Synthèse de peptides et de phosphopeptides via une ligature assistée par phosphate
WO2014194361A1 (fr) * 2013-06-04 2014-12-11 The University Of Sydney Ligature peptidique

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8765677B2 (en) * 2009-09-21 2014-07-01 The University Of Queensland Omega conotoxin peptides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5948418A (en) * 1996-03-29 1999-09-07 Estee Lauder, Inc. Sulfur-based amides and bis-amides useful against skin disorders
WO2010111737A1 (fr) * 2009-03-31 2010-10-07 The University Of Sydney Synthèse de peptides et de phosphopeptides via une ligature assistée par phosphate
WO2014194361A1 (fr) * 2013-06-04 2014-12-11 The University Of Sydney Ligature peptidique

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
A GHASSEMIAN ET AL., BIOORGANIC AND MEDICINAL CHEMISTRY, vol. 21, 2013, pages 3473 - 3478
CHEN, L. ET AL.: "A novel universal method for preparation of phenylselenyl esters", TRANSACTIONS OF TIANJIN UNIVERSITY, vol. 2, no. 1, 1996, pages 81 - 85, XP055480712 *
DUREK, T. ET AL.: "Preformed selenoesters enable rapid native chemical ligation at intractable sites", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 50, 2011, pages 12042 - 12045, XP055480714 *
GHOSH, S. K. ET AL.: "A one-pot peptide synthesis via Se-phenyl carboselenoate in mixed aqueous/organic solvent system", BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, vol. 66, 1993, pages 1566 - 1568, XP009505815 *
L R MALINS ET AL., JOURNAL OF PEPTIDE SCIENCE, vol. 20, 2013, pages 64 - 77
MALINS, L. R. ET AL.: "Modern extensions of native chemical ligation for chemical protein synthesis", PROTEIN LIGATION AND TOTAL SYNTHESIS I, vol. 362, 2015, pages 27 - 88, XP009505865, [retrieved on 20141218] *
MALINS, L. R. ET AL.: "Oxidative deselenization of selenocysteine: Applications for programmed ligation at serine", ANGEWANDTE CHEMIE, vol. 127, 2015, pages 12907 - 12912, XP009505813 *
MALINS, L. R. ET AL.: "Peptide ligation chemistry at selenol amino acids", JOURNAL OF PEPTIDE SCIENCE, vol. 20, 2014, pages 64 - 77, XP055480703, [retrieved on 20131128] *
MALINS, L. R. ET AL.: "Recent extensions to native chemical ligation for the chemical synthesis of peptides and proteins", CURRENT OPINION IN CHEMICAL BIOLOGY, vol. 22, 2014, pages 70 - 78, XP055480707, [retrieved on 20141003] *
MALINS, L. R. ET AL.: "Synthesis and utility of beta-selenolphenylalanine for native chemical ligation-deselenization chemistry", ORGANIC LETTERS, vol. 14, no. 12, 2012, pages 3142 - 3145, XP055480705 *
MITCHELL, N. J. ET AL.: "Rapid additive-free selenocystine-selenoester peptide ligation", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 137, 2015, pages 14011 - 14014, XP055480721 *
See also references of EP3265469A4 *
T DUREKP F ALEWOOD, ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 50, 2011, pages 12042 - 12045

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114249794A (zh) * 2021-12-22 2022-03-29 深圳瑞德林生物技术有限公司 一种氧化型谷胱甘肽的合成方法
CN114249794B (zh) * 2021-12-22 2023-06-23 深圳瑞德林生物技术有限公司 一种氧化型谷胱甘肽的合成方法

Also Published As

Publication number Publication date
EP3265469A1 (fr) 2018-01-10
EP3265469A4 (fr) 2019-03-06

Similar Documents

Publication Publication Date Title
Kang et al. Peptide and protein thioester synthesis via N→ S acyl transfer
CZ20022740A3 (cs) Sloučenina, způsob přípravy sloučeniny, farmaceutický prostředek a způsob léčení rakoviny
Shigenaga et al. Sequential native chemical ligation utilizing peptide thioacids derived from newly developed Fmoc-based synthetic method
US20040138412A1 (en) Extended native chemical ligation
Chen et al. Synthetic strategies for polypeptides and proteins by chemical ligation
JP2008150393A (ja) 拡張天然型化学的ライゲーション
EP3265469A1 (fr) Nouveaux procédés de synthèse
Katritzky et al. The chemical ligation of selectively S-acylated cysteine peptides to form native peptides via 5-, 11-and 14-membered cyclic transition states
EP2768842B1 (fr) Procédé de synthèse de protéines
US6686443B1 (en) Chemical reagents for formation of disulfide bonds in peptides
US20050261473A1 (en) Carboxy protection strategies for acidic c-terminal amino acids in chemical ligation of oligopeptides
US9023957B2 (en) Compound for use in peptide synthesis
AU2001288937A1 (en) Extended native chemical ligation
EP1904515B1 (fr) Ligation etendue de chaine laterale
Pineda-Castañeda et al. Designing Short Peptides: A Sisyphean Task?
JP7321537B2 (ja) ネイティブケミカルライゲーションを用いる新規フロー合成方法
JP7281826B2 (ja) ペプチドチオエステル、ペプチドの新規製造方法
Harpaz Basenlabile Auxiliare für die Erweiterung der Native Chemische Ligation in Lösung und an der Festphase
Harpaz Basenlabile Auxiliare für die Erweiterung der Native Chemische Ligation in Lösung und an der Festphase
Yang Development of novel chemical ligation methods for the synthesis of posttranslationally modified proteins
Pasunooti Synthesis of bioactive amino acid building blocks and their applications towards the peptides and glycopeptides
Camarero et al. Fmoc-Based Synthesis of Peptide r-Thioesters Using an Aryl Hydrazine Support
Callan Towards the total synthesis of apo-neocarzinostatin
Li Chemical synthesis of proteins containing post‐translational modifications and PNA‐templated disulfide bond formation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16758380

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2016758380

Country of ref document: EP